In the this study, we provide evidence for endogenous glucocorticoids (GCs) having a pathologic role in acetaminophen-induced liver injury (AILI). Specifically, pretreatment of mice with the GC receptor (GCR) inhibitor, RU486 (mifepristrone), protected normal but not adrenalectomized mice from AILI, while pretreatment with dexamethasone, a synthetic GC, exacerbated AILI. RU486 did not affect the depletion of whole liver reduced GSH and acetaminophen-protein adduct formation nor the formation of reactive oxygen species or the depletion of mitochondrial GSH or ATP. While RU486 pretreatment also protected against halothane-induced liver injury, it exacerbated concanavalin A (ConA)- and carbon tetrachloride (CCl4)-induced liver injury, demonstrating the complexity of GC effects in different types of liver injury. In this regard, genome-wide expression analysis revealed that GC-signaling following acetaminophen treatment altered mRNA expression of numerous genes that could have a role in influencing susceptibility to AILI. Conclusion: These results suggest that numerous stressful conditions that lead to elevated levels of endogenous GCs represents a previously unappreciated risk factor for liver injury caused by acetaminophen and other drugs that is likely a consequence at least in part to the diverse biological processes regulated by GCRs in cells.